Tuberoinfundibular peptide 39 (TIP39) and Parathyroid Hormone (PTH) type-2 receptor (PTH2R) constitute a new endocrine system with conserved neuronal mRNA expression in zebrafish and mouse brain. The functions associated with the murine TIP39-PTH2R system include nociception and regulation of hypothalamic (GnRH) and pituitary hormones (ACTH, GH, and LH). Zebrafish studies have shown that TIP39 and the PTH2R are expressed early in brain development in a spatial and temporal relationship with sonic hedgehog (Shh). Furthermore, embryos which have TIR39 expression inhibited by morpholino are not viable and show severely impaired brain development. Thus, the zebrafish TIP39-PTH2R studies suggest that this system serves a crucial role in neuroendocrine development and perhaps other organs such as pancreas and heart where TIR39 and the PTH2R are expressed. Therefore, the zebrafish, Danio rerio, provides a powerful model system to study TIR39-PTH2R-regulated mechanisms during development of the central nervous system (CNS). Hence, molecular analyses are proposed to better elucidate the effects of altered zebrafish PTH2R and TIR39 transcript expression during early brain development. Three aims will be pursued, 1) Evaluation of PTH2R and TIP39 MO-injected embryos during early brain development by examining the expression of Shh, GH, ACTH, LH, and GnRH. 2) Determine the relationship between TIP39 and Shh during brain development by examining the locale of cells expressing TIP39 in embryos which have a deficiency for Shh or Shh signaling (sonic you and slow-muscle-omitted). 3) Evaluation of PTH2R and TIP39 overexpression in the brain and hypothalamus (respectively) during early brain development by examining the expression of the marker genes described above. Because the PTH2R has not been analyzed by gene knockout in any organism, these experiments will provide novel insights into the role of this new TIP39-PTH2R system in normal and perturbed embryos. This work is part of a long-term molecular developmental-genetic investigation on the mechanistic role(s) of TIP39-PTH2R system during CNS, and other organ, development.